Trypanosoma cruzi is the etiologic agent of Chagas disease in humans, a disease that afflicts over 20 million people in South and Central America. Although the disease is essentially absent from North America, T. cruzi is indigenous to portions of the southwestern United States. In contrast to the considerable body of molecular genetic research on African trypanosomes, little is known about transcription and transcriptional regulation of protein encoding genes in T. cruzi. This application describes experiments that focus on developing a better understanding of transcriptional regulation in T. cruzi through a continuation of our analysis of expression of the ubiquitin and ubiquitin associated genes. Our studies of the 2.65 and 2.8 calmodulin-ubiquitin loci of I. cruzi have- shown that these loci consists of tandemly repeated calmodulin (Cal) genes followed by a single copy of the calmodulin-ubiquitin associated (CUB) gene which is in turn followed by one of the FUS genes and one or more PUB genes. Although DNA sequence analysis has shown that these genes are encoded on the same DNA strand, nuclear run-on analysis-suggests that the calmodulin genes are a part of a separate transcription unit. Therefore it seems probable that each calmodulin-ubiquitin locus contains at least two transcription units, one terminating and the other initiating within the 5'-flanking region. To further our analysis of the expression of the CUB2.65, FUS1 and PUB12.5 genes we have used the 5'-fanking regions of each gene to develop efficient transient and stable transformation systems for 1. cruzi. As with other trypanosome species in which analogous transformation systems have been developed our ability to introduce DNA into I. cruzi and study its expression will continue to facilitate our investigations. The experiments described in this proposal use genetic methodology to identify the sequences required for transcription and trans-splicing of the tandemly arrayed CUB2.65, FUSI and PUB12.5 genes.